Oxidative Decorations of the Salvinorin A Backbone: Characterization of Salvia divinorum Cytochrome P450s
Date
2019-08-23
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Abstract
Salvia divinorum is an ethnomedicinal plant belonging to the Lamiaceae (mint) family. The main bioactive compound in S. divinorum is the psychotropic diterpenoid salvinorin A. Salvinorin A was the first naturally occurring, non-nitrogenous, human kappa opioid receptor agonist identified, it thus holds potential for the treatment of addiction and mental disorders. Heterologous production of salvinorin A is desirable as purification from S. divinorum or total synthesis remains arduous. However, only the first step in the biosynthesis of salvinorin A has been elucidated, the formation of (-)-kolavenyl diphosphate (KPP) from the universal diterpenoid precursor, geranylgeranylpyrophosphate by the class II diterpene synthase (diTPS) enzyme, SdCPS2. To date, no class I diTPS which acts upon KPP has been identified, but in Saccharomyces cerevisiae, KPP can be dephosphorylated by endogenous phosphatases to yield kolavenol (KOH), the predicted class I diTPS product. From KOH, a cytochrome P450 monooxygenase (P450) known as crotonolide G synthase (SdCS) can form crotonolide G. Additional oxidative decorations to the salvinorin A backbone are also likely catalyzed by P450s. We thus sought to identify P450s which can catalyze the decorative steps succeeding KPP/KOH and crotonolide G. Since salvinorin A accumulates in the peltate glandular trichomes of S. divinorum, we mined a trichome-specific transcriptome, identifying eight candidate P450s. Of the eight P450s tested, using S. cerevisiae as a platform for heterologous expression and compound production, two P450s, CYP728D25 and CYP728D26, utilized crotonolide G to generate distinctly oxidized compounds. The highest titre of the compounds identified was obtained by having the six pathway genes necessary to synthesize SA intermediates both integrated into the yeast genome using CRISPR-Cas9 and expressed in plasmids. These results advance diterpene biosynthesis knowledge and bring us closer to obtaining therapeutic SA through heterologous means.
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Keywords
Salvia divinorum, salvinorin A, cytochrome P450
Citation
Ngo, I. (2019). Oxidative Decorations of the Salvinorin A Backbone: Characterization of Salvia divinorum Cytochrome P450s (Master's thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.